Luterial and method for isolating and culturing the same

ABSTRACT

The present invention relates to blood-derived luterial and a method for isolating and culturing the same. The luterial according to the present invention is a cell or cell-like structure having the following characteristics: (1) it is present in body fluids, including blood, sperm, intestinal juices, saliva, and cellular fluids; (2) it shows a positive staining with Janus green B, Acridine Orange and Rhodamine 123 in an immunofluorescence test; (3) in an optimal environment (pH 7.2-7.4), it has the property of expressing the genes homologous to beta-proteobacteria and gamma-proteobacteria, and has a size of 30-800 nm; (4) in an acidic environment, it has the property of expressing not only the genes homologous to beta-proteobacteria and gamma-proteobacteria, but also eukaryote-derived genes (particularly Streptophyta gene), and grows to a size ranging from 400 nm or more to 2000 nm or more; (5) it is involved in ATP production under normal conditions; and (6) it differs from mitochondria, completely differs from exosomes, and has fusion characteristics corresponding to those of an intermediary between a prokaryote and an eukaryote.

TECHNICAL FIELD

The present invention relates to luterial which is a mitochondrial-likeunidentified nano-sized particle derived from a body fluid and to amethod for isolating and culturing the same.

BACKGROUND ART

Micro-substances such as microvesicles in blood have previously beenrecognized as substance having no special function. However, variousexperimental data have demonstrated that microvesicles also havebiological activity. For example, it was found that platelet-derivedmicrovesicles function to stimulate certain cells through vesicularsurface proteins (CD154, RANTES and/or PF-4; Thromb. Haemost.(1999)82:794; J. Boil. Chem. (1999) 274:7545), and it was reported thatphysiologically active lipids (e.g., HTET or arachidonic acid) inplatelet microvesicles have certain effects on certain target cells (J.Biol. Chem.(2001) 276;19672; Cardiovasc. Res. (2001) 49(5):88). Thus,because the characteristics (e.g., size, surface antigens, determinationof cell-of-origin, payload) of substances such as vesicles present inbiological samples, can provide a diagnostic, prognostic or theranosticreadout, there remains a need to identify biological markers that can beused to detect and treat disease. Accordingly, there has been an attemptto use RNA and other biological markers associated with vesicles as wellas the characteristics of vesicles to provide a diagnosis, prognosis, ortheranosis (see WO 2011/127219).

Meanwhile, cancer is a disease in which cells grow abnormally tointerfere with the functions of normal cells, and typical examplesthereof include lung cancer, gastric cancer (GC), breast cancer (BRC),colorectal cancer (CRC) and the like, but cancer can actually occur inany tissue. In the past, the diagnosis of cancer was based on theexternal change of biological tissue caused by the growth of cancercells, but in recent years, it has been attempted to perform diagnosisand detection using trace biomolecules (glycol chain, DNA, etc.) presentin blood, biological tissue or cells. However, the cancer diagnosticmethod that is most commonly used is a diagnostic method that useseither a tissue sample obtained through biopsy or imaging. Biopsy,however, causes great pain in the patient, is costly, and requires along time for diagnosis of cancer. In addition, if a patient has cancer,the cancer can metastasize during biopsy, and in the case of a site fromwhich a tissue sample cannot be obtained through biopsy, there is adisadvantage in that the diagnosis of disease is impossible before atissue suspected of having the disease is extracted by a surgicaloperation. Meanwhile, in diagnosis based on imaging, cancer is diagnosedbased on X-ray imaging, nuclear magnetic resonance (NMR) imagingemploying an imaging agent having a disease-targeting agent attachedthereto, or the like. However, this imaging-based diagnostic method hasdisadvantages in that an erroneous diagnosis may result from the lowskill of a clinical physician or an interpreting physician and in thatthe method greatly depends on the precision of an imaging device.Furthermore, the imaging-based diagnostic method has a disadvantage inthat it is difficult to detect disease in an early stage, because eventhe most precise device cannot detect a tumor having a size of severalmm or less. In addition, the imaging-based diagnostic method hasdisadvantages in that, because a patient or a person suspected of havingdisease is exposed to high-energy electromagnetic waves for imaging,which can cause a genetic mutation, the method can cause anotherdisease, and in that the number of diagnoses by imaging is limited.

In other words, biopsy for cancer diagnosis is time-consuming, costly,inconvenient, and causes pain. For this reason, there is a need for amethod capable of significantly reducing the number of unnecessarybiopsy procedures, as well as a method capable of diagnosing cancer atan early stage.

Under such circumstances, the present inventors found that a disease canbe diagnosed and predicted by observing the characteristics of amicro-substance present in a body fluid discharged from a patient. Thecontent of this finding was filed for a patent on Jul. 12, 2013 (KoreanPatent Application No. 10-2013-0082060). The present inventors named theunidentified nano-sized particle a “luterial”.

However, a technology of efficiently isolating and culturing themicro-substance luterial so as to be capable of being clinically appliedhas not been known.

Accordingly, the present inventors have developed a method capable ofeffectively isolating the unidentified nano-sized particle luterialpresent in a body fluid discharged from a patient or a normal person andhave characterized luterial isolated by this method, thereby completingthe present invention.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a method forisolating and culturing luterial present in a body fluid discharged froma patient or a normal person.

Another object of the present invention is to provide luterial which hasintegrative characteristics corresponding to those of an intermediarybetween a prokaryote and an eukaryote, shows a positive fluorescencestaining reaction with Janus Green B, Mitotracker Red and Rhodamine 123,is mobile, and has the ability to produce ATP.

Technical Solution

To achieve the above object, the present invention provides a method forisolating luterial, comprising the steps of: separating platelet andblood-derived substances having a size greater than that of plateletfrom blood; centrifuging the blood fraction obtained after removal ofthe platelet and the blood-derived substances having a size greater thanthat of platelet; isolating luterial by collecting a supernatant afterthe centrifugation; and (4) washing the isolated luterial.

The present invention also provides a method for isolating luterialcomprising the steps of: centrifuging a body fluid to provide asupernatant, and filtering the supernatant through a filter having apore size of 2-5 μm, thereby obtaining a filtered solution; andcentrifuging the filtered solution to provide a supernatant, andfiltering the supernatant through a filter having a pore size of 0.5-2μm.

The present invention also provides body fluid-derived luterial havingone or more of the following characteristics:

(a) it shows a positive staining reaction with Janus green B, AcridineOrange and Rhodamine 123 in a fluorescence test;

(b) in an optimal environment (pH 7.2-7.4), it expressesbeta-proteobacteria-derived and gamma-proteobacteria-derived genes andhas a size of 30-800 nm;

(c) in an acidic ennvironment, it expresses not onlybeta-proteobacteria-derived and gamma-proteobacteria-derived genes, butalso eukaryote Streptophyta genes and grows to a size of 400 nm-2000 nmor more;

(d) it is involved in ATP production in normal conditions;

(e) it is a cell or cell-like structure completely different frommitochondria or exosomes;

(f) it is circular or oval in shape in a normal status, andpatient-derived luterial has a size (long axis diameter: 800 nm or more)greater than that of normal-status luterial and is mutated to formmutant luterial having a non-uniform morphology;

(g) it has a multiple ring-like membranes and is adherent;

(h) it can be present inside or outside cells;

(i) it is mobile and undergoes fusion and/or fission events;

(j) mutant luterial bursts in a certain condition and has stemness afterbursting; and

(k) it has a function of regulating p53 gene and telomeres.

The present invention also provides a method for culturing luterial,comprising: adding water to the isolated body fluid-derived luterial;and culturing the luterial at a temperature of 18 to 30° C. underirradiation with IR light.

The present invention also provides an anticancer composite containingluterial as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of the blood-derived unidentified nano-sizedparticle luterial imaged with a confocal laser scanning microscope(Zeiss), a transmission electron microscope, a scanning electronmicroscope, an atomic force microscope and a confocal scanner (LeicaTCS-SP8).

FIG. 2 depicts images showing the shape or morphology of luterial withvarious sizes ((a): 39.6-49.0 nm, an ultra-high resolution microscope(SR-GSD) image after staining with Mito-tracker Red; (b): 50.1-85.1 nm,an ultra-high resolution microscope (SR-GSD) image after staining withMito-tracker Red; (c): 76.5 nm, a transmission electron microscopeimage; (d): 160 nm, a transmission electron microscope image; (e):170-230 nm, a transmission electron microscope image, amultiple-membrane structure; (f): 234 nm, an image after staining withJanus green B; (g): 250 nm, an atomic force microscope image; (h): 361nm, a transmission electron microscope image; (i): 650.1 nm, atransmission electron microscope image; and (j): a laser scanningmicroscope image of luterial having a size of 5 μm or more afterstaining with DAPI (4′,6-diamidino-2-phenylindole).

FIG. 3 is an image showing the results of staining luterial withRhodamine 123 and then observing whether the luterial would bepositively stained.

FIG. 4 is an image showing the results of staining luterial withMito-tracker and then observing whether the luterial would be positivelystained.

FIG. 5 is an image showing the results of staining luterial withAcridine Orange and then observing whether the luterial would bepositively stained.

FIG. 6 is an image showing the results of staining luterial with DAPI(4′,6-diamidino-2-phenylindole) and then observing whether the luterialwould be positively stained.

FIG. 7 depicts images showing the results of measuring the mobility ofluterial using nano-trackers ((a) before measurement; (b) after 1second; (c) after 3 seconds).

FIG. 8 shows life cycling A of normal luterial and life cycling B ofmutated luterial.

FIG. 9 shows the life cycling and characteristics of mutated luterial.

FIG. 10 shows luterial isolated from the cancer patient body fluid.Specifically, FIG. 10(a) shows cancer patient-derived luterial whileforming elongated branches, and FIG. 10(b) shows cancer patient-derivedluterial stained with DAPI (4′,6-diamidino-2-phenylindole), Mito-trackerand Rhodamine 123.

FIG. 11 shows the life cycle of luterial.

FIG. 12 is an image showing the results of measuring the sizes ofluterial and mutated luterial.

FIG. 13 depicts images showing the results of scratching luterial withan atomic force microscope probe and removing the membrane.

FIGS. 14(a) and 14(b) are atomic force microscope images of mutatedluterials that are in a fusion status, and FIGS. 14(c) and 14(d) showthe results of imaging the mutated luterials with an atomic forcemicroscope after peeling off the membrane with a cantilever.

FIG. 15 depicts images showing the results of scratching luterial withan atomic force microscope probe, and then observing DNA through aDAPI-stained image.

FIG. 16(a) shows the bioanalyzer results of analyzing whether luterialcontains DNA.

FIG. 16(b) shows the results of qRT-PCR, which indicate that the GAPDHgene expression of DNA changes depending on the size of luterial.

FIG. 17 shows the bioanalyzer results of analyzing whether luterialcontains DNA.

FIG. 18 shows the results of measuring ATP content in media havingdifferent luterials added thereto by use of a luciferin-luciferasereaction and a luminometer (SSH: three-star ring; SSF: fisetin; 12 h:activated at 37° C. for 12 hours before an experiment).

FIG. 19 is a photograph showing a difference between luterial andexosome. In FIG. 19, the exosome has a size of 20-120 nm, an unclearmembrane and a relatively light internal color, and the luterial has asize of 50-800 nm and a distinct membrane or a packed internalstructure.

FIG. 20 depicts photographs comparing the morphology between luterial,exosome and microvesicle.

FIG. 21 depicts transmission electron microscope (TEM) images of aluterial library described in an example of the present invention.

FIG. 22 depicts confocal laser scanning microscope images showing thechange in size of luterial caused by culture.

FIG. 23 depicts images showing the change in morphology and size ofluterial caused by culture.

FIG. 24 shows percentage of bacterial homology of luterial DNA asdetermined by 16S rRNA sequencing of luterials having various sizes,derived from the blood of healthy persons (blood pH: 7.2-7.4) ((a): 100nm or less; (b): 100-200 nm; (c): 200-400 nm; (d): 400-800 nm).

FIG. 25 shows percentage of bacterial homology of luterial DNA asdetermined by 16S rRNA sequencing of luterials having various sizes,derived from blood and sperm which are in a fatigue and disease status(pH: 7.0 or less) ((a): 100 nm or less; (b): 100-200 nm; (c): 200-400nm; and (d): 400-800 nm).

FIGS. 26(a), 26(b) and 26(c) show phylogenetic trees based on the 16SrRNA sequence of blood-derived luterials.

FIG. 27 shows cell viability measured by an MTT assay after treatingovarian cancer cell lines (SKOV3 and A2780) with varying concentrationsof luterials having a size of 100-800 nm and the commercially availableanticancer drug cisplatin.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a skilled expert in thefield to which the invention pertains. Generally, the nomenclature usedherein and the experiment methods, which will be described below, arethose well known and commonly employed in the the technical field towhich the invention pertains.

As used herein, the term “luterial” named by the present inventorsrefers to a living organism present in animals and means a finesubstance having a size ranging from a size similar to that of virus toabout 800 nm (50-800 nm at normal fission stage/800 nm or more atabnormal fusion stage). Luterial has the following characteristics: (1)it is a cell or cell-like structure having integrative characteristicscorresponding to those of an intermediary between a prokaryote and aneukaryote; (2) it is present in body fluids, including blood, sperm,intestinal juices, saliva, cellular fluids, etc.; (3) it shows apositive staining reaction with Janus green B, Acridine Orange andRhodamine 123 in afluorescence staining test; (4) in an optimalenvironment (pH 7.2-7.4), it has the property of expressing geneshomologous to beta-proteobacteria and gamma-proteobacteria and has asize of 30-800 nm; (5) in an acidic environment, it expresses not onlygenes homologous to beta-proteobacteria and gamma-proteobacteria, butalso eukaryotic genes (particularly Streptophyta genes), and grows to asize of 400-2000 nm or more; (6) it is involved in ATP production undernormal conditions; and (7) it is a cell or cell-like structure whichdiffers from mitochondria and completely differs from exosomes. In thecase of mammals (including humans), luterial is present in blood,saliva, lymphatic ducts, sperm, vaginal fluids, mother's milk(particularly colostrums), umbilical cord blood, brain cells, spinalcords, and marrow. In addition, in the case of horned animals, luterialis also present in horns.

Normal luterials have a size of 50-800 nm, and mutant luterials formedby fusion have a size of a few tens of micrometers. The term “luterial”may refer to proto mitochondria containing mRNA, miRNA and DNA. Luterialis unique in that it does not dissolve in digestive fluid andinfiltrates into blood.

It is expected that luterial will be closely associated with not onlysignal transduction, cell differentiation and cell death, but also theregulation of cell cycling and cell growth. The present inventors havefound that luterial is closely associated with the diagnosis of cancer.

Normal luterial is expected to function to prevent the growth of cancercells and return cells to a healthy immune state, and the functionsthereof are performed by its RNAi (RNA interference) activity that worksto normalize genes. When an information system in RNA in the blood ofhealthy people or animals deviates from a normal status and directs toproduce a protein that causes an abnormal disease, luterial willdeliberately interfere with the information system so as to inhibit thedevelopment of diseases such as cancer. When luterial grows to a size of200-500 nm or more, it will also be involved in energy metabolism, andwhen luterial is irradiated with light having a certain wavelength, itwill function to amplify light energy in response and will act likechlorophyll. Thus, if luterial does not perform normal functions, it cancause a serious disorder in homeostasis and ATP production and can causediseases in both respiration and energy metabolism.

Mutant luterials that cannot perform normal functions as described aboveshow phenomena and characteristics different from those of normalluterials and have various sizes or shapes. Specifically, normalluterials ceases to grow after they form double spores, but mutantluterials that are found in the blood of cancer patients or patientswith chronic diseases have the property of growing infinitely, similarto stem cells, and thus have a size ranging from 600-800 nm to 200 μm(200,000 nm) or even bigger. In addition, similarly to viruses,luterials show unique characteristics that could enter and grow insideerythrocytes, leukocytes, platelets or the like or aggregate with otherluterials.

Thus, it is expected that diseases can be diagnosed or treated byobserving the morphological or biochemical characteristics of luterialand thereby promising its wide use in countless applications. However,luterial isolated from body fluids discharged from animals (includinghumans) is difficult to observe as it quickly disintegrates in vitro orundergoes morphological changes. Furthermore even the normal luterial ischanged into mutant luterial within 24 hours under an abnormalenvironment, making it difficult to accurately diagnose or treatdiseases.

In the present invention, the unidentified nano-sized particle luterialpresent in body fluids isolated from patients or normal people wasisolated by two methods.

Therefore, in one aspect, the present invention is directed to a methodfor isolating luterial from a body fluid.

A first method according to the present invention is a method forisolating luterial from blood, comprising the steps of: (1) separatingplatelet and blood-derived substances having a size greater than that ofplatelet from blood; (2) centrifuging the blood after the removal ofplatelet and the blood-derived substances having a size greater thanthat of platelet; (3) isolating luterial from a resultant supernatantobtained from centrifugation; and (4) washing the isolated luterial.Step (1) may comprise a step of passing the blood through a filterhaving a pore size of 0.8-1.2 μm and removing unfiltered substances.Step (2) may comprise a step of repeatedly centrifuging the blood at1,200-5,000 rpm for 5-10 minutes to remove general microvesicles such asexosomes and recovering the supernatant. Step (3) may comprise a step ofirradiating visible light to the supernatant obtained by thecentrifugation and isolating mobile luterial particles, which aregathered toward light, by pipetting. The blood used in step (1) may bederived from humans among mammals. Luterial is autofluorescent andmobile, and thus luterial particles in the supernatant can be isolatedby pipetting the visualized luterial under a dark-field microscope or aconfocal microscope with the assistance of irradiation of visible light.Luterial isolated in step (3) may be passed through a filter having apore size of 50 nm, and an unfiltered portion may be washed out with PBSfor isolation of luterial. Because luterial has a long axis diameter of50 nm or more, blood-derived substances smaller than luterial can beremoved by the above-described procedure.

A second method according to the present invention is a method forisolating luterial from a body fluid such as blood or sperm, comprisingthe steps of: centrifuging the body fluid to provide a supernatant, andfiltering the supernatant through a filter having a pore size of 2-5 μm,thereby obtaining a filtered solution; and centrifuging the filteredsolution to provide a supernatant, and filtering the supernatant througha filter having a pore size of 0.5-2 μm.

Specifically, the second method may comprise the steps of: centrifugingthe body fluid at 2,000-4,000 rpm for 5-30 minutes to provide asupernatant, and filtering the supernatant through a filter having apore size of 2-5 μm; and centrifuging the filtered solution at3,000-7,000 rpm for 5-20 minutes, followed by filtration through afilter having a pore size of 0.5-2 μm.

The second method may further comprise a step of irradiating visiblelight to the filtered solution and isolating mobile luterial particles,which are gathered toward the light, by pipetting. Herein, the luterialis autofluorescent and mobile, and thus luterial particles in thesupernatant can be visualized by irradiation with visible light. Theisolated luterial may be passed through a filter having a pore size of50 nm, and an unfiltered portion may be washed out with PBS, therebyobtaining luterial. Because luterial has a long axis diameter of 50 nmor more, blood-derived substances smaller than luterial can be removedby the above-described procedure.

The luterial isolated by each of such two methods can be observed by adark-field microscope or a confocal microscope, and can be dividedaccording to size into 50-200 nm (developmental phase)/ 200-400 nm(maturation phase)/400-600 nm (mitosis phase)/ 600-800 nm (over-mitosisphase) by sequential use of 200 nm, 400 nm, 600 nm, 800 nm and 1000 nmfilters.

In the present invention, the isolated luterial was characterized.

(1) Morphology

It was found that normal luterials have a size of 50-800 nm (FIGS. 2 and12), and grow up to a size of 800 nm in the absence of abnormal fusion.The patient-derived luterials have a size (long axis diameter of 800 nmor more) greater than that of healthy person-derived luterials, aremutated to form mutant luterials having a non-uniform morphology, andgrow to a size of several thousands of nm when abnormal fusion persists.

In addition, luterial is circular or oval in shape, and shows a multiplering-like membrane structure in SEM or TEM images, but had no internalcristae structure (FIG. 1).

(2) Fluorescent Staining

It is known that mitochondria are positively stained by Janus green Band fluorescent dyes, including Rhodamine 123, Mitotracker, AcridineOrange, and DAPI, and it was found that luterial is also stained by thesame dyes as those for mitochondria. Fluorescence images indicated thatthe luterial, but not exosomes, showed a reaction similar to that ofmitochondria in fluorescent staining test and showed autofluorescence(FIGS. 2(a), 2(b), 2(f) and 2(j), and FIGS. 3 to 6).

(3) Properties

Unlike exosomes and microvesicles, luterials were adherent and mobileand underwent fusion or fission events. It was found that mutantluterial did burst under certain conditions, had sternness afterbursting, and could be present inside or outside cells (FIGS. 8, 9 and11).

(4) ATP Production

ATP production in luterial having a size of 200-400 nm was demonstratedusing luciferin-luciferase reaction and a luminometer. A media containngluterial showed an increase in ATP concentration compared to a mediawithout luterial, indicating that luterial has the ability to produceATP. SSH and SSF were further added to the media and their effects onATP production by luterial were examined.A mediacontaining SSF inducedhigher ATP production by luterialcompared to a mediawith SSH, thusfinding a medium mix that is capable of efficiently increasing the ATPproduction by luterial (FIG. 18).

(5) Content of Nucleic Acids

It was found by DAPI and acridine orange (AO) staining that luterialcontains not only RNA, but also DNA. Specifically, AO is known to stainRNA with orange AOat an excitation wavelength of 460 nm and an emissionwavelength of 650 nm, and DNA with green at an excitation wavelength of502 nm and an emission wavelength of 525 nm. DAPI is known to positivelystain for DNA. Luterial according to the present invention was confirmedto contain RNA and DNA using the staining test as described above (FIGS.5 and 6). RNA in luterial were further isolated and purified using anextraction kit, and then subjected to agarose gel electrophoresis afterqRT-PCR against human GAPDH gene transcripts. It was found that theexpression level of human GAPDH gene changed depending on the size ofthe luterial (FIGS. 2(h), 16 and 17).

(6) 16S rRNA Sequencing

The gDNA of luterial was extracted using a FastDNA SPIN Kit (MPBiomedicals, Cat 6560-200), and then the 16S rRNA gene was amplifiedusing the primers shown in Tables 1 and 2 below.

In addition, the 1461 amplified gene fragments were analyzed for theirhomology using GenBank database (NCBI database). As a result, luterialsderived from blood and sperm showed 16S rRNA sequences having homologywith those of genes derived from β-proteobacteria, γ-proteobacteria,Acidobacteria, Cyanobacteria, Actinobacteria, Firmicutes and eukaryotes,and showed integrative characteristics corresponding to those of anintermediary between a prokaryote and an eukaryote (FIGS. 24 and 25).

It was observed that, in an optimal condition (blood pH: 7.2-7.4),blood-derived luterial showed homology with genes derived fromβ-proteobacteria and γ-proteobacteria (FIG. 24) and had a size of 50-800nm.

In normal conditions, sperm-derived luterial showed homology with genesderived from β-Proteobacteria and γ-Proteobacteria, Bacteroidetes andChordata.

On the contrary, in an acidic condition, not only genes derived fromβ-proteobacteria and γ-proteobacteria as in normal conditions, but alsoother diverse bacteria-derived genes and eukaryote-derived genes wereexpressed, . The luterial mainly expressed the 16S rRNA characteristicsof Streptophyta and planctomy (FIG. 25) and grew to a size of 400-2000nm.

TABLE 1 Forward primers Sequence (Adaptor-key- SEQ ID Taxon Namelinker-target sequence) NOs: Bacteria B16S-F 5′-CCTATCCCCTGTGTGCCTTG 1GCAGTC-TCAG-AC-GAGTTTGA TCMTGGCTCAG-3′ Bifido- Bif16S-F5′-CCTATCCCCTGTGTGCCTTG 2 bacterium GCAGTC-TCAG-AC-GGGTTCGATTCTGGCTCAG-3′

TABLE 2 Reverse primers SEQ Sequence (Adaptor-key- ID Taxon Namelinker-target sequence) NO: Bacteria B16-7-4 5′-CCATCTCATCCCTGCGTGTC  3TCCGAC-TCAG-AGAGCTG-AC- WTTACCGCGGCTGCTGG-3′ Bacteria B16-7-75′-CCATCTCATCCCTGCGTGTC  4 TCCGAC-TCAG-TCAGATG-AC- WTTACCGCGGCTGCTGG-3′Bacteria B16-7-8 5′-CCATCTCATCCCTGCGTGTC  5 TCCGAC-TCAG-CGATGAG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-7-12 5′-CCATCTCATCCCTGCGTGTC  6TCCGAC-TCAG-TCTGCAG-AC- WTTACCGCGGCTGCTGG-3′ Bacteria B16-7-135′-CCATCTCATCCCTGCGTGTC  7 TCCGAC-TCAG-AGCGATG-AC- WTTACCGCGGCTGCTGG-3′Bacteria B16-8-3 5′-CCATCTCATCCCTGCGTGTC  8 TCCGAC-TCAG-ATGCTGAG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-8-4 5′-CCATCTCATCCCTGCGTGTC  9TCCGAC-TCAG-TACAGCAG-AC- WTTACCGCGGCTGCTGG-3′ Bacteria B16-8-185′-CCATCTCATCCCTGCGTGTC 10 TCCGAC-TCAG-ATCGTGTG-AC- WTTACCGCGGCTGCTGG-3′Bacteria B16-8-21 5′-CCATCTCATCCCTGCGTGTC 11 TCCGAC-TCAG-CTACACAG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-9-4 5′-CCATCTCATCCCTGCGTGTC 12TCCGAC-TCAG-CGTGTACTG- AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-9-55′-CCATCTCATCCCTGCGTGTC 13 TCCGAC-TCAG-CTGTCTACG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-9-8 5′-CCATCTCATCCCTGCGTGTC 14TCCGAC-TCAG-AGTCACTAG- AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-9-125′-CCATCTCATCCCTGCGTGTC 15 TCCGAC-TCAG-AGCTCACTG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-10-6 5′-CCATCTCATCCCTGCGTGTC 16TCCGAC-TCAG-ATCACGTGCG- AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-10-75′-CCATCTCATCCCTGCGTGTC 17 TCCGAC-TCAG-ATAGCTCTCG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-10-8 5′-CCATCTCATCCCTGCGTGTC 18TCCGAC-TCAG-AGTGAGCTCG- AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-10-95′-CCATCTCATCCCTGCGTGTC 19 TCCGAC-TCAG-AGTCTGACTG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-11-1 5′-CCATCTCATCCCTGCGTGTC 20TCCGAC-TCAG-TCATATACGCG- AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-11-25′-CCATCTCATCCCTGCGTGTC 21 TCCGAC-TCAG-TAGATAGTGCG-AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-11-3 5′-CCATCTCATCCCTGCGTGTC 22TCCGAC-TCAG-ACGTCTCTACG- AC-WTTACCGCGGCTGCTGG-3′ Bacteria B16-11-45′-CCATCTCATCCCTGCGTGTC 23 TCCGAC-TCAG-CTAGAGACACT-AC-WTTACCGCGGCTGCTGG-3′

(7) Differences from Exosomes and Mitochondria

Table 3 below summarizes the differences of luterials from exosomes andmitochondria.

TABLE 3 No. Category Exosomes Luterials Mitochondria 1 Size 20~120 nm50~800 nm 400~1,000 nm 2 Fluorescence (CD63antibody)GFP+(CD63antibody)GFP− (CD63antibody)GFP− 3 Mitotracker Red− MitotrackerRed+ Mitotracker Red+ 4 Janus Green B− Janus Green B+ Janus Green B+ 5Rhodamine 123− Rhodamine 123+ Rhodamine 123+ 6 Mobility − 13~25 μm/sec −7 Growth in Culture − + − 8 Natural Growth − + − 9 ATP Synthesis − + +10 Auto-fluorescence − + N/A 11 Fusion + + + 12 Kiss-and-run − + +(Fission and Fusion) 13 Sequencing 18SrRNA 16SrRNA 16srRNA 28S rRNA(GammaProtebacteria Alpha Proteobacteria Beta ProteobacteriaBacteroidetes 14 Habitat Out of cell In-and-out of Cell In cell

Luterials have an average size of 200-800 nm, which is smaller than thatof mitochondria (400-1,000 nm) and greater than that of exosomes (20-120nm), and exosomes have unclear membranes and a relatively light internalcolor, whereas luterials have distinct membranes or a packed internalstructure (FIG. 19). In addition, luterials have a morphology completelydifferent from those of exosomes and microvesicles (FIG. 20).

In fluorescent staining, luterials unlike exosomes show a reactionsimilar to that of mitochondria. Luterials are present inside andoutside of cells while exosomes are present outside of cells only.Luterials can be supplied by taking foods, whereas mitochondria areintracellular substances that cannot be provided by intake of foods.

In addition unlike exosomes and mitochondria, luterials are mobile, andcan grow naturally, the growth thereof can be maintained by culture, andshow autofluorescence. Furthermore, luterials, exosomes and mitochondriaall undergo fusion events, but in exosomes, kiss-and-run motion and ATPproduction are absent. Moreover, exosomes are present outside the cellsand mitochondria are present inside the cells, whereas luterials can bepresent inside or outside the cells (FIG. 11).

The results of 16S rRNA sequencing indicated that mitochondria showedhomology with a-proteobacteria, whereas luterials showed homology withγ-proteobacteria, β-proteobacteria, Bacteroidetes, Firmicutes andeukaryotes.

In another aspect, the present invention is focused on the bodyfluid-derived luterial having one or more of the followingcharacteristics:

(a) it shows a positive staining with Janus green B, Acridine Orange andRhodamine 123 in a fluorescence test;

(b) in an optimal environment (pH 7.2-7.4), it expresses geneshomologous to beta-proteobacteria and gamma-proteobacteria, and has asize of 30-800 nm;

(c) in an acidic environment, it expresses genes homologous to not onlybeta-proteobacteria and gamma-proteobacteria, but also eukaryoteStreptophyta, and grows to a size of 400 nm-2000 nm or more;

(d) it is involved in ATP production in normal conditions;

(e) it is a cell or cell-like structure completely different frommitochondria or exosomes;

(f) it is circular or oval in shape in a normal condition, andpatient-derived luterial has a size (long axis diameter: 800 nm or more)greater than that of normal luterial and is mutated to form mutantluterial having a non-uniform morphology;

(g) it has a multiple ring-like membrane structure and is adherent;

(h) it can be present inside or outside cells;

(i) it is mobile and undergoes fusion and/or fission events;

(j) mutant luterial bursts in a certain condition and has stemness afterbursting; and

(k) it has a function of regulating p53 gene and telomeres.

Meanwhile, the size (diameter), area, morphology and nano-tracking speedof luterial differ depending on the presence or absence of disease in anindividual, and thus one or more of the above-described characteristicsmake it possible to diagnose disease or predict disease prognosis. Thiscan be seen from the fact that luterial derived from a healthy personhaving no disease and luterial derived from a person having disease havedifferent sizes, morphologies, nano tracking speeds, etc.

Normal luterials in healthy persons merely form double spores undergoingfission, but luterials (mutant luterials) in patients with chronicdisease or cancer have characteristics in that they fuse or coagulatewith one another or burst to adhere to cells such as erythrocytes orcancer cells, thereby changing their morphology and size abnormally(FIGS. 8 to 10). Mutant luterials are highly adherent, and thus thefusion thereof is accelerated by the above-described cycle to increasetheir size to about 600-800 nm or more, and any of such mutant luterialsmay also have a size of 200 μm (200,000 nm) or more. The presentinventors found that the morphology of luterials is consistent dependingon the kind or progress of cancer, and the content of this finding wasfiled for a patent (Korean Patent Application No. 10-2013-0082060).

Thus, it is possible to diagnose disease or predict disease prognosis byobserving the morphological or biochemical characteristics of luterials,indicating that luterials can be used in unlimited applications.

Luterials have a normal form, a flagellum form, a mass form, a rod form,or a combination form. Herein, the normal form may be a form that doesnot undergo additional modification such as fusion or bursting, with along axis diameter-to-short axis diameter ratio of 1:1-3:1. Theluterials can show a shape close to a circular shape. They appear assmall spots in microscopic observation.

The flagellum form may be a form that results from the modification orfusion of luterials to have flagella-like structure attached outside.The present inventors found that the percentage of the flagellum formdramatically increased as cancer progresses to terminal cancer and thatthis flagellum form luterials were observed in 99.1% of the patientsdiagnosed as stage 4 cancer (Korean Patent Application No.2013-0082060). If the percentage of the flagellum-form luterial reaches80-100%, it would work as a tumor marker to indicate a terminal stagecancer. The survival period of such patients is about 1-4 months, andparticularly, patients dominated with flagellum form luterials cannotsurvive for a long period.

The mass form (M shape) is a form that was changed from the normal formdue to the bursting or fusion of luterials. It is an irregular bulkyshape whose long axis diameter-to-short axis diameter ratio is notgreat. Preferably, it may have a long axis diameter-to-short axisdiameter ratio of 3:1-5:1. Various forms of the mass form are observed.

The rod form (R shape) refers to a formresulting from the bursting,modification or fusion of luterials. It has a long axisdiameter-to-short axis diameter ratio greater than that of the massshape. Preferably, it may have a long axis diameter-to-short axisdiameter ratio of 5:1-12:1. It includes a rod 1 form consisted ofcircular or oval single chains; and a rod 2 form consisted of two ormore single chains bonded to one or another. The rod 1 form refers tothe single luterial that has grown to a rod shape. It may result fromthe bursting and/or mutation. The rod 2 form refers to a rod shapedluterial formed from fusion of two or more luterials. It may result fromone or more of bursting, mutation and fusion. The flagellum form may beincluded in a broad sense in the scope of the rod form, but it would bedifferent from the rod form in that it has a flagellum-like structure.Thus, luterial form should be first determined whether it is of the rodform and then depending on the presence of the flagellum-like structureit should be further categorized into the flagellum form.

The combination form may be a combination of the rod shape and the massshape. It may mean that a portion of a single micro particulate matterhas the rod shape and the other portion thereof has the mass shape.

The rod form may be one selected from the group consisting of: a rod 1form consisted of a single circular or oval shape; and a rod 2 formconsisted of two or more single chains bonded to one another. Thecombination form may be a combination of the rod shape and the massshape.

As described above, the morphology of luterials in vivo changesdepending on the development and progress of disease, and thus it ispossible to diagnose disease or predict disease prognosis by observingthe morphological characteristics of luterials. In addition, themorphological change of luterials is also associated with changes in thecontent of nucleic acids in the luterials and the sequence of theluterials, and thus enabling diagnosis of disease from nucleic acidexpression pattern analysis (16S rRNA sequencing) of luterials. Forexample, it is possible to diagnose disease (particularly cancer) bycomparing the 16S rRNA sequence of normal luterial with that ofpatient-derived luterial. Particularly, co-expression of Streptopytagene and eukaryote gene can be used as a marker for diagnosing andpredicting carcinogenesis.

However, luterials isolated from body fluids discharged from patients ornormal people are difficult to observe because they tend to disappear invitro within a short time or change their shape. In addition, in anabnormal environment, normal luterials are changed into mutant luterialswithin 24 hours, making it difficult to accurately diagnose or treatdiseases. However, according to the culture method of the presentinvention, luterials can be cultured such that the their size do notexceed certain size (500 nm).

Therefore, in another aspect, the present invention is directed to amethod for culturing luterial, comprising: adding water to luterial; andculturing the luterial at a temperature of 18 to 30° C. (preferably 20to 25° C.) under irradiation with IR light. The water that is added inthe culture process may be saline or PBS solution, but is not limitedthereto. The body fluid-derived luterial before culture may be obtainedaccording to the isolation method of the present invention and may havea size of 50-200 nm. The luterial cultured according to the culturemethod of the present invention may have a size of 300-800 nm. Herein,the luterial can be controlled to a size of 500 nm or less undermicroscopic observation. After completion of the culture, the luterialmay be sorted according to size, and cooled and stored at −80° C. orstored under nitrogen or may also be stored at a temperature above zero.For storage, a preservative may be added to the luterial.

The luterial cultured as described above can be stored for a certainperiod of time without changing the characteristics of the luterial, andcan be effectively used to diagnose disease and predict diseaseprognosis. As used herein, the expression “without changing thecharacteristics of luterial” means that the morphology or size ofluterial is maintained at a level similar to that before culture inmedia. In addition, it means that the activity of luterial, such asmobility (e.g., nano-tracking speed), is maintained at a value similarto that before culture.

Specifically, luterial cultured according to the culture method of thepresent invention may be used for the following purposes. Mutantluterials have an abnormally increased morphology or size due to fusionor coagulation, unlike normal luterials (FIGS. 8 to 10). By culturingthe mutant luterials, a substance capable of inhibiting or preventingthe mutation of luterials can be screened from the candidate substancesby observing whether the changes in cultured luterial mutants.

Furthermore, a substance that promotes the fission of mutant luterialscan be screened by treating cultured mutant luterials with a candidatesubstance or means. Since mutant luterials show patterns of fusion orcoagulation events (FIGS. 8, 9 and 11), by treating the mutant luterialswith a candidate substance and examining whether it promotes the fissionof mutant luterials to have the size of normal luterials, it would bepossible to screen for a substance that inhibits the mutation ofluterials or converts mutant luterials to normal luterials, that is, asubstance that prevents disease caused from mutant luterials.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are illustrative purposesonly and are not to be construed to limit the scope of the presentinvention.

Thus, the substantial scope of the present invention will be defined bythe appended claims and equivalents thereof.

Example 1 Isolation of Blood-Derived Luterials

50 cc of blood was collected from a non-small cell lung cancer patientand passed through a filter having a pore size of 0.8 μm or more, andunfiltered substances were removed. The filtered blood was repeatedlycentrifuged at 1,200-5,000 rpm for 5-10 minutes to remove generalmicrovesicles such as exosomes collected in pellets, and the supernatantwas collected. The supernatant was irradiated with visible light, andthe gathered luterial particles with mobility were isolated bypipetting. Because luterial is autofluorescent and mobile, luterialparticles could be visualized by irradiation with visible light asdescribed above. At this time, mobile luterial particles were isolatedby pipetting under observation with a dark-field microscope or aconfocal microscope. The isolated luterials were filtered through afilter having a pore size of 50 nm, and only an unfiltered portion waswashed with PBS, thereby obtaining luterials. According to the aboveprocedures, luterials having a long axis diameter of 50-800 nm could beobtained, which could be observed through a dark-field microscope or aconfocal microscope. The obtained luterials were sorted according tosize into 50-200 nm (developmental phase)/200-400 nm (maturation phase)/400-600 nm (mitosis phase)/600-800nm (over-mitosis phase). According toa similar method, a library of luterials with various sizes as shown inFIG. 21 was constructed, and the morphologies of luterials with varioussizes are shown in FIG. 2.

Example 2 Isolation of Semen-Derived Luterials

Semen was centrifuged at 2000-4000 rpm for 5-30 minutes, and thesupernatant was filtered through a filter having a pore size of 2-5 μm.The filtered solution was centrifuged at 3000-7000 rpm for 5-20 minutes,followed by filtration through a filter having a pore size of 0.5-2 μm.Because luterials are autofluorescent and mobile, luterial particles canbe visualized when the filtered solution was irradiated with visiblelight. At this time, mobile luterial particles were isolated bypipetting under observation with a dark-field microscope or a confocalmicroscope. The isolated luterials were filtered through a filter havinga pore size of 50 nm, and only an unfiltered portion was washed withPBS, thereby obtaining luterials which could be observed through adark-field microscope or a confocal microscope.

Example 3 Characteristics of Luterials

(1) Structure

Among the luterials obtained in Example 1, luterials having a size ofabout 50-400 nm were imaged with a confocal laser scanning microscope(Zeiss), a transmission electron microscope, a scanning electronmicroscope, an atomic force microscope and a confocal scanner (LeicaTCS-SP8). As a result, it was shown that the luterials also had amultiple ring-like layers of membrane structure and a non-completedinternal cristae structure, similar to mitochondria, and were observedin the same wavelength range as that for mitochondria. In addition, itcould be observed that the luterials were circular or oval in shape(FIGS. 1, 1(e), 2(h), 13 and 14).

(2) Staining Characteristics

Among the luterials obtained in Example 1, luterials having a size ofabout 50-800 nm were stained with Mito-tracker, Rhodamine 123, AcridineOrange and Janus green B, and tested for their positive staining. Theresults showed that even the plant-derived luterials were also stainedby Mito-tracker, Rhodamine 123, Acridine Orange and Janus green B (FIGS.2(a), 2(b), 2(f) and 2(j), and FIGS. 3 to 6).

(3) Autofluorescence

Among the luterials obtained in Example 1, luterials having a size ofabout 50-800 nm were analyzed through fluorescence images. The resultshowed that luterials responded to light (FIG. 5).

(4) Mobility

The mobility of luterials obtained in Example 1 was measured bynano-tracking (3i Inc., USA) analysis. Specifically, luterials wereobserved with a bright-field microscope, tracking was set in the centerof the luterial, and nano-tracking was performed. Then, the real-timemovement trajectory of the luterial was recorded and the speed persecond of the luterial was calculated (FIG. 7).

As a result, the nano-tracking speed of the luterials according to thepresent invention was measured to be about 13-25 μm/sec.

(5) Analysis of Whether Luterials Contain RNA and DNA

The luterials having a size of 200-400 nm, isolated in Example 1, wereimaged with an atomic force microscope. As shown in FIGS. 2(h), 15, 16and 17, luterials contain nucleic acids such as RNA or DNA.

In order to isolate total RNA and DNA from luterials having a size of200-400 nm isolated in Example 1, a QIAGEN kit (RNeasy Micro Kit: Cat74004)was used, followed by quantification using an Experion RNA (DNA)StdSens (Bio-Rad) chip.

Luterials were recovered by centrifugation (at 8,000 g for 1 hr 30 min),and then lysed by adding 50 μl of lysis buffer RLT plus (guanidineisothiocycanate, detergents) from the kit mixed with 3.5 μl ofbeta-mercaptoethanol and then passing them 5-10 times through a syringeequipped with a 20-gauge needle. The sample lysis buffer was thentransferred to an AllPrep DNA spin column, followed by centrifugation(at ≧8000 g for 15 sec) to separate DNA remaining on the column from theRNAcontained in the buffer that passed through the column.

Next, 350 μl of 70% ethanol was added to the same volume of the samplelysis buffer that passed through the column (RNA) and well mixed. Then700 μl of the mixture was transferred to a RNease MinElute spin columnand centrifuged (at ≧8000 g for 15 sec), and the buffer that passedthrough the column was removed. The column was washed sequentially with350 μl of RW1, 500 μl of RPE buffer and 500 μl of 80% ethanol. All thecentrifugation procedures (≧8000 g for 15 sec) used as described abovewere performed under the same conditions. To obtain RNA, 14 μl ofRNeasy-free solution was added to the column and then centrifuged (at≧8000 g for 60 sec), thereby isolating luterial RNA.

For isolation of genomic DNA (gDNA), a FastDNA SPIN Kit (MP Biomedical)was used. The isolated luterials were added to the tube, followed byaddition of 978 μl of sodium phosphate buffer and 122 μl of MT buffer.The mixture was homogenized for 40 sec, and then centrifuged at 14,000 gfor 10 min to collect the supernatant, after which 250 μl of PPS(Protein Precipitation Solution) was added to the supernatant and mixedfor 10 min. After centrifugation at 14,000 g for 5 min, and thesupernatant was transferred into a 15 ml tube, and this procedure wasrepeated twice. For DNA binding, the resulting supernatant was placed ona rotor for 2 minutes, and then placed on a silica matrix support for 3minutes. 600 μl of the supernatant was carefully added to a SPIN filterand was centrifuged at 14,000g for 1 min, and then the supernatant wasdiscarded, and 500 μl of SEWS-M was added to the remaining pellets andresuspended. After centrifugation for 1 min, the supernatant wasdiscarded, and centrifugation was repeated such that any buffer wouldnot remain. Then, 50 μl of DES (DNase/Pyrogen-Free Water) was added tothe remaining material, followed by centrifugation at 14,000g for 1 min,and then genomic DNA was isolated.

Quantification was performed using an Experion RNA (DNA) StdSens(Bio-Rad) chip. The result, as shown in FIGS. 16 and 17, indicated thatthe luterials contained RNA and DNA.

(6) 16S rRNA Sequencing

16S rRNA (ribosomal ribonucleic acid) is a DNA that interacts withvarious proteins to form ribosomes. Because the rate of change in thenucleotide sequence of 16S rRNA is significantly lower than those of thenucleotide sequences of most of other genes in the genome, it isrecognized that the similarity of the 16S rRNA sequence reflects thephylogenetic distance between organisms.

{circle around (1)} Blood-Derived Luterials

The luterials obtained in Example 1 were treated using a FastDNA SPINkit (MP Biomedicals, Cat 6560-200) to extract gDNA. Using the extractedgDNA, the 16S rRNA of the luterial was amplified using a PCR-premix(iNtRON Biotechnology, Korea) and primers of SEQ ID NOs: 1 to 23.

The amplified PCR products were sequenced using a BigDye TerminatorCycle Sequencing Ready Reaction kit (Applied Biosystems, USA) and anautomated DNA analyzer system (PRISM 3730XL DNA analyzer, AppliedBiosystems). The amplified PCR products were a total of 1461 fragments.Among them, 1407 fragments showed homology with theproteobacteria-derived gene, 20 fragments showed homology with theAcidobacteria-derived gene, and 11 fragments showed homology with theActinobacteria-derived gene (Table 4).

The fragments with the analyzed nucleotide sequences were combined usingSeqMan software (DNASTAR), thereby obtaining the nucleotide sequence of16S rRNA.

FIG. 24 shows bacterial homologies of luterial DNA of healthy individualas determined by 16S rRNA sequencing of luterials derived from the bloodof healthy persons (blood pH: 7.2-7.4), and shows the results ofanalysis performed for luterials of various sizes ((a): 100 nm or less,(b): 100-200 nm, (c) 200-400 nm, and (d) 400-800 nm). There was nosignificant difference among the luterial sizes, and luterials allshowed homology with the genes derived from Proteobacteria, Firmicutesand Bacteroidetes.

FIG. 25(c) shows bacterial homologies of luterial DNA obtained from thepatients with disease. The 16S rRNA sequencing data of 200-400 nmluterials derived from blood of a patient with a fatigue or diseasecondition (blood pH: 7.0 or less) were used. Unlike in healthyconditions, luterial genes obtained from the patients showed homologywith Streptophyta-derived genes.

FIGS. 26(a) 26(b) and 26(c) show phylogenetic trees based on the 16SrRNA sequence of blood-derived luterials.

TABLE 4 SUM Sum Rank Taxonomy Name LKL-B (Ratio) LKL-B (Number) PhylumBacteria;;;Proteobacteria Proteobacteria 96.3039 96.3039 1407 1407Phylum Bacteria;;;Acidobacteria Acidobacteria 1.36893 1.36893 20 20Phylum Bacteria;;;Actinobacteria Actinobacteria 0.75291 0.75291 11 11Phylum Bacteria;;;Bacteroidetes Bacteroidetes 0.54757 0.54757 8 8 PhylumBacteria;;;Cyanobacteria Cyanobacteria 0.41068 0.41068 6 6 PhylumEukarya;Viridiplantae;;Streptophyta Streptophyta 0.27379 0.27379 4 4Phylum Bacteria;;;Firmicutes Firmicutes 0.20534 0.20534 3 3 PhylumBacteria;;;TM6 TM6 0.06845 0.06845 1 1 Phylum Bacteria;;;PlanctomycetesPlanctomycetes 0.06845 0.06845 1 1

It was shown that the 16S rRNA fragments of the blood-derived luterialsshowed homology with various bacteria, including beta-proteobacteria,gamma-proteobacteria, Bacteroidetes, Firmicutes and Streptophyta.

Generally, when the relatedness of gDNA in microbialtaxonomy is lessthan 70%, the microorganisms are recognized as independent strains. Inaddition, it was demonstrated by statistical analysis that, when thehomology of the 16S rRNA sequence is less than 97%, the gDNA relatednessis less than 70%. Thus, cells having a homology of 97.0% or more withthe 16S rRNA fragments of the luterials were analyzed. As shown inTables 5 to 7 below, the blood-derived luterials showed a homology of100% with gamma-proteobacteria, a homology of 97.53% with Firmicutes,and a homology of 97% or more with Bacteroidetes.

Meanwhile, as shown in Table 8, abnormal acidic luterials showed ahomology of 99% or more with Streptophyta.

TABLE 5 Characteristics of Luterial by 16S rRNA Seq Raw data Hit Simi-Taxonomic Seq Name Sequence accession larity assignment IOFBYRO01DTJ3IATTGAACGCTGGCGGCAGGCTTAACACA AM410704 100 Bacteria;;;Proteobacteria;;TGCAAGTCGAGCGGAGATGAGGTGCTTG Gammaproteobacteria;;CACCTTATCTTAGCGGCGGACGGTGAGT Pseudomonadales;;AATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGGACAACATTCCGAAAGGGATGCTAAT Acinetobacter; ACCGCATACGTCCTACGGGAGAAAGCAGAcinetobacter junii;; GGGATCTCCGGACCTTGCGCTAATAGAT LMG 998-AM410704(T)GAGCCTAAGTCGGATTAGCTAGTTGGTG GGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCA CACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG GACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGG TTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTGAGACTAATACTCTTGGATAGTGG ACGTTACTCGCAGAATAAGCACCGGCTA ACTCTGTGIOFBYRO01DUOG5 ATTGAACGCTGGCGGCAGGCTTAACACA AM410704 100Bacteria;;;Proteobacteria;; TGCAAGTCGAGCGGAGATGAGGTGCTTGGammaproteobacteria;; CACCTTATCTTAGCGGCGGACGGGTGAG Pseudomonadales;;TAATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGACAACATTCCGAAAGGAATGCTAATA Acinetobacter; CCGCATACGTCCTACGGGAGAAAGCAGGAcinetobacter junii;; GGATCTTCGGACCTTGCGCTAATAGATG LMG 998-AM410704(T)AGCCTAAGTCGGATTAGCTAGTTGGTGG GGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCAC ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG GACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGG TTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTGAGACTAATACTCTTGGATAGTGG ACGTTACTCGCAGAATAAGCACCGGCTA ACTCTGTGIOFBYRO01BHYT4 TTGAACGCTGGCGGCAGGCTTAACACAT AM410704 100Bacteria;;;Proteobacteria;; GCAAGTCGAGCGGAGATGAGGTGCTTGGammaproteobacteria;; CACCTTATCTTAGCGGCGGACGGGTGAG Pseudomonadales;;TAATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGACAACATTCCGAAAGGGAATGCTAAT Acinetobacter;AcinetobacterACCGCATACGTCCTACGGGGAGAAAGCA junii;; GGGGATCTTCGGACCTTGCGCTAATAGALMG 998-AM410704(T) TGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGAT CTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGCGGGGAATATTGGACAATGGGGGGAACCCTGATCCAG CCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGG AGGCTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGG CTAACTCTGTG IOFBYRO01CYAL1ATTGAACGCTGGCGGCAGGCTTAACACA AM410704 100 Bacteria;;;Proteobacteria;;TGCAAGTCGAGCGGAGATGAGGTGCTTG Gammaproteobacteria;;CACCTTATCTTAGCGGCGGACGGGTGAG Pseudomonadales;;TAATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGACAACATTCCGAAAGGAATGCTAATA Acinetobacter;AcinetobacterCCGCATACGTCCTACGGGAGAAAGCAGG junii;; GGATCTTCGGACCTTGCGCTAATAGATGLMG 998-AM410704(T) AGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTG TAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCCTGATCCAGCC ATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAG GCTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCT AACTCTGTG IOFBYRO01DRDH1ATTGAACGCTGGCGGCAGGCTTAACACA AM410704 100 Bacteria;;;Proteobacteria;;TGCAAGTCGAGCGGAGATGAGGTGCTTG Gammaproteobacteria;;CACCTTATCTTAGCGGCGGACGGGTGAG Pseudomonadales;;TAATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGACAACATTCCGAAAGGAATGCTAACA Acinetobacter;AcinetobacterCCGCATACGTCCTACGGGAGAAAGCAGG junii;; GGATCTTCGGACCTTGCGCTAATAGATGLMG 998-AM410704(T) AGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTG TAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCA TGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGG CTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTA ACTCTGTG IOFBYRO01BWKSQATTGAACGCTGGCGGCAGGCTTAACACA AM410704 100 Bacteria;;;Proteobacteria;;TGCAAGTCGAGCGGAGATGAGGTGCTTG Gammaproteobacteria;;CACCTTATCTTAGCGGCGGACGGGTGAG Pseudomonadales;;TAATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGACAACATTCCGAAAGGAATGCTAATA Acinetobacter;AcinetobacterCCGCATACGTCCTACGGGAGAAAGCAGG junii;; GGATCTTCGGACCTTGCGCTAATAGATGLMG 998-AM410704(T) AGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTG TAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCA TGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGG CTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTA ACTCTGTG IOFBYRO01BWKSOATTGAACGCTGGCGGCAGGCTTAACACA AM410704 100 Bacteria;;;Proteobacteria;;TGCAAGTCGAGCGGAGATGAGGTGCTTG Gammaproteobacteria;;CACCTTATCTTAGCGGCGGACGGGTGAG Pseudomonadales;;TAATGCTTAGGAATCTGCCTATTAGTGGG Moraxellaceae;;GGACAACATTCCGAAAGGAATGCTAATA Acinetobacter;AcinetobacterCCGCATACGTCCTACGGGAGAAAGCAGG junii;; GGATCTTCGGACCTTGCGCTAATAGATGLMG 998-AM410704(T) AGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTG TAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCA TGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGG CTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTA ACTCTGTG IOFBYRO01A8KAWTATTAGTGGGGGACAACATTCCGAAAGG  AKIQ01000085 100Bacteria;;;Proteobacteria;; AATGCTAATCCGCATACGTCCTACGGGAGammaproteobacteria;; GAAAGCAGGGGACCTTCGGGCCTTGCGC Pseudomonadales;;TAATAGATGAGCCTAAGTCGGATTAGCT Moraxellaceae;;AGTTGGTGGGGTAAAGGCCTACCAAGGC Acinetobacter;AcinetobacterGACGATCTGTAGCGGGTCTGAGAGGATG venetianus;;RAG-1-ATCCGCCACACTGGGACTGAGACACGGC AKIQ01000085(T) CCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTG ATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGA GGAGGAGGCTACTAGTATTAATACTACTGGATAGTGGACGTTACTCGCAGAATAAGC ACCGGCTAACTCTGTG

TABLE 6 Firmicutes Raw data Hit Simi- Taxonomic Seq Name Sequenceaccession larity assignment IOFBYRO01ANZSO GGCGGCGTGCCTAATACATGCAAGTAGAADVN01000004 97.53 Bacteria;;Firmicutes;; ACGCTGAAGCTTGGTGCTTGCACCGAGCBacilli;;Lactobacillales;; GGATGAGTTGCGAACGGGTGAGTAACGCStreptococcaceae;; GTAGGTAACCTGCCTCTTAGCGGGGGATStreptococcus;Streptococcus AACTATTGGAAACGATAGCTAATACAGCAparasanguinis;;ATCC 15912- TAAAAGTCGATATCGCATGATATTGATTT ADVN01000004(T)GAAAGGTGCAATTGCATCACTAAGAGAT GGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATAC ATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTC CTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCA ACGCCGCGTGAGTGAAGAAGGTTTTTCGGATCGTAAAGCTCTGTTGTAAGAGAAGAA CGAGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAACTTACCAGAAAGGGACGGC TAACTACGTG

TABLE 7 Bacteroidetes Raw data Hit Simi- Taxonomic Seq Name Sequenceaccession larity assignment IOFBYRO01BUV34 TGAACGCTAGCGGCAGGCTTAATACATG4P004046 99.79       Bacteria;;;Bacteroidetes;;CAAGTCGTGGGGCAGCACAGAATAGCAA Sphingobacteria;;TATTTGGGTGGCGACCGGCAAACGGGTG Sphingobacteriales;;CGGAACACGTACACAACCTTCCGATAAG Chitinophagaceae;;TGGGGGATAGCCCAGAGAAATTTGGATT 4P004046_g;4P004046_s;;AATACCCCGTAACATATAGAGATGGCATC 4P004046 GTCTTTATATTATAGCTTCGGTGCTTATTGATGGGTGTGCGTCTGATTAGGTAGTTG GCGGGGTAACGGCCCACCAAGCCTACGATCAGTAGCTGATGTGAGAGCATGATCA GCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAGCAGTAAGGAA TATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAAGGATGAATGTCC TCTGGATTGTAAACTTCTTTTATTTGGGACGAAAAAGAGCATTCTTGCTCACTTGACG GTACCAAGTGAATAAGCACCGGCTAACT CCGTGIOFBYRO01AEZDS GATGAACGCTAGCGATAGGCCTAACACA FJ672469 97.34      Bacteria;;;Bacteroidetes;; TGCAAGTCGAGGGGCAGCACATGAAGTABacteroidia;;Bacteroidales;; GCAATACTGATGGTGGCGACCGGCGCAPorphyromonadaceae;; CGGGTGAGTAACACGTATGCAACCTACCAB243818_g;FJ672469_s;; TTCAACAGGAGAATAACCCGTCGAAAGA FJ672469CGGACTAATACTCCATAACACAGGGATC CCACATGGGAATATTTGTTAAAGATTTATCGGTTGAAGATGGGCATGCGCTCCATTA GCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGGATAGGGGAACTGAGAG GTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCA GTGAGGAATATTGGTCAATGGAGGCAACTCTGAACCAGCCACGTCGCGTGAAGGAT GACGGCCCTACGGGTTGTAAACTTCTTTTGTAAGGGAATAAAGTTAGTTACGTGTAAC TATTTGCATGTACCTTACGAATAAGGATCGGCTAACTCCGTG IOFBYRO01BP52Z GATGAACGCTAGCGATAGGCCTAACACA FJ67246997.34       Bacteria;;;Bacteroidetes;; TGCAAGTCGAGGGGCAGCACATGAAGTABacteroidia;;Bacteroidales;; GCAATACTGATGGTGGCGACCGGCGCAPorphyromonadaceae;; CGGGTGAGTAACACGTATGCAACCTACCAB243818_g;FJ672469_s;; TTCAACAGGAGAATAACCCGTCGAAAGA FJ672469CGGACTAATACTCCATAACACAGGGATC CCACATGGGAATATTTGTTAAAGATTTATCGGTTGAAGATGGGCATGCGCTCCATTA GCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGGATAGGGGAACTGAGAG GTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCA GTGAGGAATATTGGTCAATGGAGGCAACTCTGAACCAGCCACGTCGCGTGAAGGAT GACGGCCCTACGGGTTGTAAACTTCTTTTGTAAGGGAATAAAGTTAGTTACGTGTAAC TATTTGCATGTACCTTACGAATAAGGATCGGCTAACTCCGTG IOFBYRO01BBMIP TGAACGCTAGCGGCAGGCTTAATACATG 4P00404699.79       Bacteria;;;Bacteroidetes;; CAAGTCGTGGGGCAGCACAGAATAGCAASphingobacteria;; TATTTGGGTGGCGACCGGCAAACGGGTG Sphingobacteriales;;CGGAACACGTACACAACCTTCCGATAAG Chitinophagaceae;;TGGGGGATAGCCCAGAGAAATTTGGATT 4P004046_g;4P004046_s;;AATACCCCGTAACATATAGAGATGGCATC 4P004046 GTCTTTATATTATAGCTTCGGTGCTTATTGATGGGTGTGCGTCTGATTAGGTAGTTG GCGGGGTAACGGCCCACCAAGCCTACGATCAGTAGCTGATGTGAGAGCATGATCA GCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAGCAGTAAGGAA TATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAAGGATGAATGTCC TCTGGATTGTAAACTTCTTTTATTTGGGACGAAAAAAGAGCATTCTTGCTCACTTGAC GGTACCAAGTGAATAAGCACCGGCTAAC TCCGTGIOFBYRO01BBHTW ATGGACGCTAGCGGCAGGCTTAATACAT 4P004046 99.58      Bacteria;;;Bacteroidetes;; GCAAGTCGTGGGGCAGCACAGAATAGCASphingobacteria;; ATATTGGGTGGCGACCGGCAAACGGGT Sphingobacteriales;;GCGGAACACGTACACAACCTTCCGATAA Chitinophagaceae;;GTGGGGGATAGCCCAGAGAAATTTGGAT 4P004046_g;4P004046_s;;TAATACCCCGTAACATATAGAGATGGCAT 4P004046 CGTCTTTATATTATAGCTTCGGCGCTTATTGATGGGTGTGCGTCTAATTAGGTAGTT GGCGGGGTAACGGCCCACCAAGCCTACGATCAGTAGCTGATGTGAGAGCATGATC AGCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAGCAGTAAGG AATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAAGGATGAATGT CCTCTGGATTGTAAACTTCTTTTATTTGGGACGAAAAAAGAGCATTCTTGCTCACTTG ACGGTACCAAGTGAATAAGCACCGGCTA ACTCCGTGIOFBYRO01BQCEI GATGAACGCTAGCGATAGGCCTAACACA FJ672469 97.34      Bacteria;;;Bacteroidetes;; TGCAAGTCGAAGGGGCAGCACATGAAGTBacteroidia;;Bacteroidales;; AGCAATACTGATGGTGGCGACCGGCGCAPorphyromonadaceae;; CGGGTGAGTAACACGTATGCAACCTACCAB243818_g;FJ672469_s;; TTCAACAGGAGAATAACCCGTCGAAAGA FJ672469CGGACTAATACTCCATAACACAGGGATC CCACATGGGAATATTTGTTAAAGAGTTTATCGGTTGAAGATGGGCATGCGCTCCATT AGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGGATAGGGGAACTGAGA GGTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGC AGTGAGGAATATTGGTCAATGGAGGCAACTCTGAACCAGCCACGTCGCGTGAAGGA TGACGGCCCTACGGGTTGTAAACTTCTTTTGTAAGGGAATAAAGTTAGTTACGTGTAA CTATTTGCATGTACCTTACGAATAAGGATCGGCTAACTCCGTG IOFBYRO01CGIIX ATGAACGCTAGCGGCAGGCTTAATACAT FN66565997.8        Bacteria;;;Bacteroidetes;; GCAAGTCGAGGGGCAGCACGGTATAGCSphingobacteria;; AATATATGGGTGGCGACCGGCAAACGGG Sphingobacteriales;;TGCGGAACACGTACACAACCTTCCGGTG Chitinophagaceae;;AGCGGGGGATAGCCCAGAGAAATTTGGA Hydrotalea;Hydrotalea TTAATACCCCATACTATAATGATCAGGCA flava;; TCTGGTTATTATCAAAGGCTTCGGCCGCTCCUG 51397-FN665659(T) TATTGATGGGTGTGCGTCTGATTAGGTAGTTGGCGGGGTAGAGGCCCACCAAGCC TACGATCAGTAGCTGATGTGAGAGCATGATCAGCCACACGGGCACTGAGACACGGG CCCGACTCCTACGGGAGGCAGCAGTAAGGAATATTGGACAATGGACGCAAGTCTG ATCCAGCCATGCTGCGTGAAGGATGAATGCCCTCTGGGTTGTAAACTTCTTTTACAG GGGAAGAAAGTTATCTTTTTTAGGATATTTGACGGTACCCTATGAATAAGCACCGGC TAACTCCGTG IOFBYRO01BUV35TGAACGCTAGCGGCAGGCTTAATACATG 4P004047 99.24906689Bacteria;;;Bacteroidetes;; CAAGTCGTGGGGCAGCACAGAATAGCAASphingobacteria;; TATTTGGGTGGCGACCGGCAAACGGGTG Sphingobacteriales;;CGGAACACGTACACAACCTTCCGATAAG Chitinophagaceae;;TGGGGGATAGCCCAGAGAAATTTGGATT 4P004046_g;4P004046_s;;AATACCCCGTAACATATAGAGATGGCATC 4P004047 GTCTTTATATTATAGCTTCGGTGCTTATTGATGGGTGTGCGTCTGATTAGGTAGTTG GCGGGGTAACGGCCCACCAAGCCTACGATCAGTAGCTGATGTGAGAGCATGATCA GCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAGCAGTAAGGAA TATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAAGGATGAATGTCC TCTGGATTGTAAACTTCTTTTATTTGGGACGAAAAAGAGCATTCTTGCTCACTTGACG GTACCAAGTGAATAAGCACCGGCTAACT CCGTG

TABLE 8 Streptophyta Raw data Hit Simi- Seq Name Sequence accessionlarity Taxonomic assignment IOFBYRO01BVMU5 GATGAACGCTGGCGGCATGCTTAACACACAAP02016081 100    Eukarya;Viridiplantae;; TGCAAGTCGGACGGGAAGTGGTGTTTCCStreptophyta;; AGTGGCGGACGGGTGAGTAACGCGTAA eudicotyledons;;coreGAACCTGCCCTTGGGAGGGGAACAACA eudicotyledons;;GCTGGAAACGGCTGCTAATACCCCGTAG Vitaceae;;Vitis;VitisGCTGAGGAGCAAAAGGAGGAATCCGCC vinifera;;CAAP02016081CGAGGAGGGGCTCGCGTCTGATTAGCTA GTTGGTGAGGCAATAGCTTACCAAGGCGATGATCAGTAGCTGGTCCGAGAGGATGA TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG GAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAG GCCCACGGGTCGTGAACTTCTTTTCCCGGAGAAGAAGCAATGACGGTATCTGGGGA ATAAGCATCGGCTAACTCTGTG IOFBYRO01DG9Y3GATGAACGCTGGCGGCATGCTTAACACA CAAP02016081 100    Eukarya;Viridiplantae;;TGCAAGTCGGACGGGAAGTGGTGTTTCC Streptophyta;; AGTGGCGGACGGGTGAGTAACGCGTAAeudicotyledons;;core GAACCTGCCCTTGGGAGGGGAACAACA eudicotyledons;;GCTGGAAACGGCTGCTAATACCCCGTAG Vitaceae;;Vitis;VitisGCTGAGGAGCAAAAGGAGGAATCCGCC vinifera;;CAAP02016081CGAGGAGGGGCTCGCGTCTGATTAGCTA GTTGGTGAGGCAATAGCTTACCAAGGCGATGATCAGTAGCTGGTCCGAGAGGATGA TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG GAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAG GCCCACGGGTCGTGAACTTCTTTTCCCGGAGAAGAAGCAATGACGGTATCTGGGGA ATAAGCATCGGCTAACTCTGTG IOFBYRO01BVXH2GATGAACGCTGGCGGCATGCTTAACACA CAAP02016081 100    Eukarya;Viridiplantae;;TGCAAGTCGGACGGGAAGTGGTGTTTCC Streptophyta;; AGTGGCGGACGGGTGAGTAACGCGTAAeudieotyledons;;core GAACCTGCCCTTGGGAGGGGAACAACA eudieotyledons;;GCTGGAAACGGCTGCTAATACCCCGTAG Vitaceae;;Vitis;VitisGCTGAGGAGCAAAAGGAGGAATCCGCC vinifera;;CAAP02016081CGAGGAGGGGCTCGCGTCTGATTAGCTA GTTGGTGAGGCAATAGCTTACCAAGGCGATGATCAGTAGCTGGTCCGAGAGGATGA TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG GAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAG GCCCACGGGTCGTGAACTTCTTTTCCCGGAGAAGAAGCAATGACGGTATCTGGGGA ATAAGCATCGGCTAACTCTGTG IOFBYRO01CVD3EGATGAACGCTGGCGGCATGCTTAACACA CAAP02016081  99.77 Eukarya;Viridiplantae;;TGCAAGTCGGACGGGAAGTGGTGTTTCC Streptophyta;; AGTGGCGGACGGGTGAGTAACGCGTAAeudieotyledons;;core GAACCTGCCCTTGGGAGGGGAACAACA eudieotyledons;;GCTGGAAACGGCTGCTAATACCCCGTAG Vitaceae;;Vitis;VitisGCTGAGGAGCAAAAGGAGGAATCCGCC vinifera;;CAAP02016081CGAGGAGGGGCTCGCGTCTGATTAGCTA GTTGGTGGGGCAATAGCTTACCAAGGCGATGATCAGTAGCTGGTCCGAGAGGATGA TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG GAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAG GCCCACGGGTCGTGAACTTCTTTTCCCGGAGAAGAAGCAATGACGGTATCTGGGGA ATAAGCATCGGCTAACTCTGTG

{circle around (2)} Semen-Derived Luterials

The semen-derived luterials obtained in Example 2 were subjected to gDNAextraction, PCR amplification and sequencing according to theabove-described method. FIG. 25 shows bacterial homology of luterial DNAas determined by 16S rRNA sequencing of luterials derived from semen inboth normal condition and a fatigue and disease condition (sperm pH: 7.0or less). The analysis was performed with the luterials of various sizes((a): 100 nm or less, (b): 100-200 nm, and (d) 400-800 nm).

The normal semen-derived luterials showed homology with the genesderived from Proteobacteria, Firmicutes and Bacteroidetes, like theblood-derived luterials. Particularly, the luterials showed homologywith the Chordata-derived gene.

Luterial DNA derived from semen in abnormal acidic conditions showedhomology with the Streptophyta-derived gene.

(7) Measurement of ATP Content

10 mL of each of four media, including a control, luterial, luterialwith SSH (12 hr) and luterial with SSF (12 hr), was placed in a tube,and glucose (100 mg/mL) and ADP substrate (1 mM) were added thereto,followed by culture in water bath at 37° C. At 30-min intervals afterthe start of the culture, 100 μl of a sample was collected, placed in atube, and diluted 10-fold with 900 μl of distilled water. Then, 10 μl ofthe sample was transferred into a fresh tube, and 100 μl of luciferasereagent contained in the ATP kit was added thereto, and measurement wasimmediately performed five times using a luminometer.

As shown in FIG. 18, the media containing luterial showed an increase inthe ATP concentration compared to the control media without luterial.Such results suggest that the luterial has the ability to produce ATP.In comparing the results between SSH and SSF media, the ATPconcentration in the S SF-added group was higher than that in theSSH-added group (FIG. 18).

Example 4 Culture of Luterials

(1) Among luterials obtained in Example 1, luterials having a size ofabout 50-200 nm were irradiated with IR light after addition of PBS, andthen cultured at 18 to 30° C. for about 3 hours. At about 1-hourintervals immediately after irradiation with IR light, the size of theluterials was measured with a microscope. After about 1-6 hours,luterials having a size of about 200 nm before culture grew to a size ofabout 500 nm. Thus, when water was added to blood-derived luterialswhich were then cultured at 18 to 30° C. under irradiation with IRlight, the luterials could grow to a size of about 500 nm. Consistently,when luterials were additionally cultured, they grew to a size ofseveral hundreds of 1 μm and did also burst during the additionalculture (FIG. 22).

(2) Among luterials obtained in Example 1, luterials having a size ofabout 400-800 nm were irradiated with IR light after addition of PBS,and then cultured at 18 to 30° C. for about 3 hours. At about 1-hourintervals immediately after irradiation with IR light, the size andstatus of luterials were measured with a microscope. After about 1-6hours, it was shown that luterials having a size of about 400-800 nmbefore culture underwent fission without growth.

In addition, it was observed that, when mutant luterials having a sizeof 800 nm or more were further cultured, they changed to mutantluterials that are seen in the blood of cancer patients (FIG. 23).

Example 5 Anticancer Effect of Luterials

In order to measure inhibitory effects of luterials on the growth of twoovarian cell lines (SKOV3 and A2780), a yellow tetrazolium MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay wasperformed. The MTT assay is a method for measuring the growth of livingcells, and is based on the principle that dehydrogenase in mitochondriaof living cells produces violet formazan when the yellow water-solublesubstance MTT is added. The production of violet formazan is known to besubstantially proportional to the number of living cells havingmetabolic activity, and thus can be very effective in measuring thegrowth and differentiation of cells.

Specifically, 100 μl of cultured cancer cells were added to a 96-wellplate at a concentration of 5×10⁴ cells/ml and cultured in a humidifiedincubator (5% carbon and 95% oxygen) at 37° C. for 24 hours, and thentreated with various concentrations of luterials having a size of100-800 nm. After 48 hours of culture, 15 μl of a solution of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well,followed by culture for 4 hours. After the formation of formazan wasconfirmed, the medium was completely removed, and 100 μl of dimethylsulfoxide (DMSO) was added to each well in order to dissolve formazanformed at the well bottom. Thereafter, the absorbance at 560 nm wasmeasured using a microplate reader (GEMINI, Stratec Biomedical), and theinhibition rate of cell growth by luterials relative to 100% of thecontrol cells was calculated.

As a result, the IC₅₀ values of luterials for the SKOV3 and A2780 celllines were 30 μg/ml and 60 μg/ml, respectively. The IC₅₀ value of thecommercially available anticancer drug cisplatin was 100 μM (FIG. 27).Luterials showed stronger cytotoxicity than the positive control drugfor the two ovarian cancer cell lines, and showed cytotoxicity similarto that of the positive control drug for normal ovarian cells.

INDUSTRIAL APPLICABILITY

As described above, according to the present invention, the unidentifiednano-sized particle luterial present in the body fluid of patients ornormal persons can be effectively isolated, and the isolated luterialcan be cultured so as to grow to a certain size. As such, luterial isuseful for the diagnosis and treatment of disease. In addition, luterialshows a strong anticancer effect against cancer cell lines, and thus isuseful as an anticancer agent.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1. A method for isolating luterial, comprising the steps of: (a)removing platelet and blood-derived substances having a size greaterthan that of platelet from blood; (b) centrifuging the blood from whichthe platelet and the blood-derived substances have been removed; (c)isolating luterial from a supernatant obtained by the centrifugation;and (d) washing the isolated luterial.
 2. The method of claim 1, whereinthe blood is derived from mammals.
 3. The method of claim 2, wherein theblood is derived from human.
 4. The method of claim 1, wherein step (a)further comprises a step of passing blood through a filter having a poresize of 0.8-1.2 μm and collecting a portion not passed through thefilter.
 5. The method of claim 4, wherein luterial is classifiedaccording to size into 50-200nm, 200-400 nm, 400-600 nm, 600-800 nm, and800-1,000 nm by the sequential use of 200 nm, 400 nm, 600 nm, 800 nm,and 1000 nm sized filters.
 6. The method of claim 1, wherein the step(b) is performed by centrifuging the blood at 1,200-5,000 rpm for 5-10minutes repeatedly.
 7. The method of claim 1, wherein exosomes areremoved in the step (b).
 8. The method of claim 1, wherein step (c) isperformed by irradiating visible light to the supernatant obtained bythe centrifugation and isolating mobile luterial particles by pipetting,which are gathered to a region where visible light is irradiated.
 9. Themethod of claim 1, wherein step (d) is performed by passing luterialisolated in step (3) through a filter having a pore size of 50 nm, andwashing out only an unfiltered portion, thereby obtaining luterial. 10.Body fluid-derived luterial having one or more of the followingcharacteristics: (a) showing a positive positive staining with Janusgreen B, Acridine Orange and Rhodamine 123 in a fluorescence test; (b)in an optimal environment (pH 7.2-7.4), expressing genes homologous tobeta-proteobacteria-derived and gamma-proteobacteria-derived genes andhas a size of 30-800 nm; (c) in an acidic environment, expressing notonly genes homologous to beta-proteobacteria-derived andgamma-proteobacteria-derived genes, but also eukaryotic Streptophytagenes and growing to a size of 400 nm-2000 nm or more; (d) involved inATP production in normal conditions; (e) a cell or cell-like structurecompletely different from mitochondria or exosomes; (f) circular or ovalin shape in a normal status, and patient-derived luterial has a size(long axis diameter: 800 nm or more) greater than that of normal-statusluterial and is mutated to form mutant luterial having a non-uniformmorphology; (g) having a membrane structure of multiple ring-like layers-and is adherent; (h) it can be present inside or outside cells; (i) itis mobile and undergoes fusion and/or fission events; (j) mutantluterial bursts in a certain condition and has stemness after bursting;and (k) it has a function of regulating p53 gene and telomeres.
 11. Thebody fluid-derived luterial of claim 10, wherein the body fluid isblood, sperm, intestinal juices, saliva, or cellular fluid derived frommammals.
 12. A method of culturing a luterial comprising adding water tothe isolated body fluid-derived luterial according to claim 10 andculturing at a temperature of 18 to 30° C. under irradiation with IRlight.
 13. The method of claim 12, wherein before and after the culture,the luterial has a size of 50-200 nm and 50-800 nm, respectively. 14.The method of claim 13, wherein the water is saline or PBS solution. 15.A method for isolating luterial, comprising the steps of: (a)centrifuging a body fluid to provide a supernatant, and filtering thesupernatant through a filter having a pore size of 2-5 μm; and (b)centrifuging the filtered solution to obtain a supernatant, andfiltering the supernatant through a filter having a pore size of 0.5-2μm.
 16. The method of claim 15, further comprising a step (c) ofirradiating visible light to the filtered solution in step (b) andisolating mobile luterial particles by pipetting those that gather to aregion where visible light is irradiated,
 17. The method of claim 15,wherein the body fluid is blood, sperm, intestinal juices, saliva, orcellular fluid derived from mammals.
 18. The method of claim 15, whereinthe centrifuging of the body fluid in step (a) is performed at2,000-4,000 rpm for 5-30 minutes.
 19. The method of claim 15, whereinthe centrifuging of the filtered solution in step (b) is performed at3,000-7,000 rpm for 5-20 minutes.
 20. An anticancer compositioncomprising the luterial of claim 10 as an active ingredient.